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Abstract:

Hydrophobic drugs become more practical for treatments by being
encapsulated in micelle compositions for increasing solubility. Micelle
compositions may include an excipient tocopherol and/or prodrug
formulations of the drug. Micelles extend the time period the drug
remains in the micelles to improve drug circulation time and thereby drug
delivery. Hydrophobic drugs for micelle encapsulation may include
rapamycin, geldanamycin, and paclitaxel. Administration of these micelle
compositions does not require Cremophor EL or Tween 80, avoiding serious
side effects associated with these products which would previously
accompany such drug administration.

Claims:

1.-22. (canceled)

23. A micelle composition comprising a plurality of micelles, wherein the
micelles comprise an amphiphilic polymer, a hydrophobic excipient with a
log Po/w greater than about 3.5 and a molecular weight less than about
1000 Da, and a hydrophobic passenger drug; wherein the hydrophobic
excipient and the hydrophobic passenger drug are located within the
micelles; and the amphiphilic linear polymer comprises a pegylated
phospholipid.

24. The micelle composition of claim 23 wherein the mol % ratio of the
hydrophobic excipient to the amphiphilic polymer is about 0.2 to about
50.

25. The micelle composition of claim 23 wherein the mol % ratio of the
hydrophobic excipient to the amphiphilic polymer is at least about 2:1.

29. The micelle composition of claim 27 wherein the concentration of the
Vitamin E is about 2 mM to about 100 mM.

30. The micelle composition of claim 29 wherein the concentration of the
Vitamin E is about 2 mM to about 20 mM.

31. The micelle composition of claim 23 wherein the hydrophobic passenger
drug is one or more of rapamycin, paclitaxel, paclitaxel prodrugs,
geldanamycin, and geldanamycin prodrugs.

32. The micelle composition of claim 31 wherein the hydrophobic passenger
drug is one or more of rapamycin, paclitaxel, or, geldanamycin.

33. The micelle composition of claim 31 wherein the hydrophobic passenger
drug is a paclitaxel prodrug comprising a carbonyloxy-linked or
silyloxy-linked prodrug moiety at one or both of the paclitaxel positions
C2 and C7, or a geldanamycin prodrug comprising a nitrogen-linked prodrug
moiety at the geldanamycin C17 in place of the C17 methoxy group; wherein
when present, the presence of the paclitaxel prodrug moiety or
geldanamycin prodrug moiety increases the octanol-water partition
coefficient log Po/w of the paclitaxel or geldanamycin prodrug compared
to paclitaxel or geldanamycin, respectively.

34. The micelle composition of claim 23 wherein the hydrophobic passenger
drug is rapamycin and the rapamycin comprises at least 11 wt. % of the
micelles.

35. The micelle composition of claim 23 wherein the hydrophobic passenger
drug is rapamycin and the concentration of rapamycin is about 0.1 mg/mL
to about 4 mg/mL.

36. The micelle composition of claim 23 wherein the amphiphilic polymer
is PEG-DSPE and the molecular weight of the PEG block is about 2 kDa.

37. The micelle composition of claim 23 wherein the hydrophobic passenger
drug is rapamycin and the CMC is about 3 μM to about 28 μM.

38. A micelle composition comprising a plurality of micelles, wherein the
micelles comprise an amphiphilic polymer, a hydrophobic excipient having
a log Po/w greater than about 3.5 and a molecular weight less than about
1000 Da, and a hydrophobic passenger drug; wherein the hydrophobic
excipient and the hydrophobic passenger drug are located within the
micelles; the amphiphilic linear polymer is a pegylated phospholipid; the
hydrophobic excipient is Vitamin E; and the hydrophobic passenger drug is
rapamycin.

39. The micelle composition of claim 38 wherein the rapamycin in the
micelles is about 10% wt. drug/wt. micelles to about 20% wt. drug/wt.
micelles.

40. A process for forming a micelle composition comprising: combining an
amphiphilic polymer, a hydrophobic excipient having a log Po/w greater
than about 3.5 and a molecular weight less than about 1000 Da, and
hydrophobic drug, in an organic solvent to form a solution; removing
substantially all of the organic solvent from the solution to leave a
substantially solvent-free mixture; and resuspending the substantially
solvent-free mixture in water or buffer, to provide the micelle
composition wherein the micelles include the hydrophobic excipient and
the hydrophobic drug in the core of the micelles.

41. The process of claim 40 wherein the concentration of the amphiphilic
polymer is about 0.1 mM to about 60 mM, the concentration of the
hydrophobic excipient is about 0.1 mM and about 600 mM, and the
concentration of the drug is about 0.1 mg/mL to about 10 mg/mL.

42. The process of claim 41 wherein the hydrophobic drug is rapamycin,
paclitaxel, or geldanamycin.

Description:

[0001] This application claims benefit of U.S. Provisional Application No.
60/670,460, filed on Apr. 12, 2005, and U.S. Provisional Application No.
60/716,000, filed on Sep. 9, 2005, which are incorporated herein by
reference.

BACKGROUND

[0003] 1. Field of the Invention

[0004] This invention is directed generally to micelle compositions,
methods of making micelles, and the use of micelle compositions with
drugs for treatment of disease.

[0005] 2. Description of the Prior Art

[0006] Cancer is a very deadly disease. Various cytoxic chemotherapy
agents have been used to eradicate cancer and/or prevent the spread of
the cancer. Alkylating agents, such as cisplatin and chlorambucil,
crosslink NDA to prevent cell division. Antitumor antibiotics, such as
dactinomycin and bleomycin, bind DNA and thus prevent DNA separation and
mRNA synthesis. Antimetabolites, such as purine and pyrimidine
antagonists and 5-fluorouracil, may mimic cell nutrients and prevent
normal DNA synthesis. Plant alkaloids, such as paclitaxel and
vinblastine, block cell division by blocking microtubule formation.
Topoisomerase inhibitors, such as camptothecins, topotecan, and
irinotecan, inhibit DNA supercoiling and block transcription and
replication. Many drugs that are potentially efficacious for treating
diseases such as cancer have poor solubility that limits their
usefulness.

[0007] Rapamycin is a large, highly hydrophobic compound with applications
in chemotherapy, immunosuppression, anti-restenosis, fungal infections,
and neurological disorders. Rapamycin as an anti-cancer agent is
generally formed as ester analogs which are quickly hydrolyzed and
sequestered into the red blood cells thereby reducing the effectiveness
of rapamycin at tumor sites. Rapamycin is currently used as an
immunosuppressant for kidney transplant patients, Rapamune
(Wyeth-Ayerst), and has shown long term clinical safety. However,
rapamycin is a poorly water soluble drug, creating difficulties in drug
administration in patients.

[0008] Geldanamycin is also a hydrophobic compound with applications
including the treatment of cancer. Geldanamycin is a member of the new
class of compounds known as heat shock protein inhibitors, having both
anti-tumor and neurological disease applications. The mode of action is
by inhibiting heat shock protein 90 (Hsp90), strongly binding to Hsp90
(Kd=1.2 μM), and preventing interaction with downstream
components. Hsp 90 is a molecular chaperon responsible for the folding,
stability, and function of numerous client proteins. Inhibition of Hsp 90
leads to the destabilization and eventual ubiquitination of many
oncogenic client proteins. By targeting multiple oncogenic proteins,
geldanamycin may be efficacious against a broad range of tumors and may
increase the chances of overcoming drug resistance. In addition, the
inhibition of Hsp90 leads to an up-regulation of Hsp70, which reduces the
formation of abnormal tau species, the primary component of plaque
deposits in Alzheimer's and Parkinson's disease.

[0009] Paclitaxel is another hydrophobic compound with applications
including the treatment of cancer. Paclitaxel belongs to a group of
medicines called antineoplastics, which inhibit cellular growth. The
inhibition is accomplished by disrupting microtubule function by binding
to the beta subunit of tubulin. The disrupted microtubule looses the
ability to disassemble, a necessary function, for example, in chromosomal
migration during cell replication. Additionally, research has indicated
that paclitaxel induces apoptosis, programmed cell death, by binding to
an apoptosis stopping protein called Bcl-2 and stopping its function.

[0010] Various techniques for solubilizing poorly soluble compounds exist,
such as the formation of emulsions, liposomes, or micelles, all of which
may have multiple phases, some of which may be unstable and may tend to
separate.

[0011] Micelle systems based on amphiphilic polymers using block
copolymers (ABC's) have been used to formulate such challenging drugs.
ABC's comprised of a hydrophobic, such as polypropylene glycol), and
hydrophilic block, such as polyethylene glycol (PEG), can assemble into a
microphase separated, core/shell architecture in a selective solvent.
PEG-poly(s-caprolactone) (PEG-PCL) and PEG-poly(amino acids) can form
these polymeric micelles. Alternatively, phospholipids can be used, such
as, PEG-distearoylphosphatidylethanolamine (PEG-DSPE) to form these
polymeric micelles. In an aqueous environment, the hydrophobic drug can
be encapsulated into the hydrophobic core of the micelle and have aqueous
solubility provided by a poly(ethylene glycol) (PEG) and corona (shell).
Due to their nanoscopic dimensions and stealth properties imparted by a
PEG corona, micelles may have long-term circulation capabilities. During
the circulation period, the micelle may gradually release drug and
eventually dissociate and be eliminated from circulation.

[0012] Excipients and co-excipients have been used to solubilize poorly
soluble compounds. Alpha-tocopherol, commonly known as Vitamin E or
simply tocopherol, has been used as an excipient because of its ring and
alkyl chain structures common to many poorly-soluble drugs. Vitamin E is
not toxic to living organisms. Additionally, tocopherol stabilizes
biological membranes. Tocopherol, however, is not soluble in water and
therefore it has had limited usefulness in intravenous solutions.

SUMMARY OF THE INVENTION

[0013] A micelle composition may comprise an amphiphilic polymer, a
hydrophobic excipient, and a hydrophobic passenger drug. In one aspect,
the amphiphilic polymer is PEG-DSPE. In another aspect, the excipient is
tocopherol. In yet another aspect, the ratio of tocopherol to PEG-DSPE is
between about 0.1 and about 3.

[0014] In one aspect, a micelle composition comprises an amphiphilic
polymer and rapamycin. In another aspect, the micelle composition may
have an amphiphilic polymer, rapamycin and tocopherol. In yet another
aspect, the concentration of PEG-DSPE may be between about 1 and about 10
mM, the concentration of tocopherol may be between about 2 and about 20
mM, and the concentration of rapamycin may be between about 0.1 and 1.0
mg/ml.

[0015] A micelle composition may comprise an amphiphilic polymer and
geldanamycin. The geldanamycin may be a geldanamycin prodrug with
increased hydrophobic properties.

[0016] A micelle composition may comprise an amphiphilic polymer and
paclitaxel. The paclitaxel may be a paclitaxel prodrug with increased
hydrophobic properties.

[0017] A process for forming micelle compositions may include mixing
amphiphilic polymer, hydrophobic excipient, and hydrophobic drug into an
organic solvent to form a solution, removing substantially all of the
organic solvent from the solution to leave a substantially solvent-free
mixture, and resuspending the solvent-free mixture in water or buffer. A
process may also include adding said solution to a substantially water
solution before removing substantially all of said organic solvent from
said solution to leave a substantially solvent-free mixture.

[0018] A process and resulting prodrug composition made for improving
micelle encapsulation efficiency of hydrophobic drugs. In anther aspect,
a process for making geldanamycin prodrugs for encapsulation. In yet
another aspect, a process for making paclitaxel prodrugs for
encapsulation.

[0019] A method of treatment for a disease or condition in a human or an
animal may comprise administering an effective amount of a micelle
composition comprising an amphiphilic polymer, a hydrophobic excipient
and a hydrophobic passenger drug.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a schematic showing a micelle structure for drug
delivery, including a hydrophobic core and a hydrophilic corona.

[0031] FIG. 12 shows a graph of critical micelle concentration at
different PEG-DSPE to tocopherol ratios as a function of the
concentration of the PEG-DSPE micelles.

[0032] FIG. 13 is a bar graph of relative core viscosity as a function of
the PEG-DSPE to tocopherol ratio.

[0033] FIG. 14 is a bar graph showing the increasing aggregate number
within the core as a function of various PEG-DSPE to tocopherol ratios.

[0034] FIG. 15 is a graph showing the stability of PEG-DSPE micelles in
phosphate buffered saline and in 4% bovine serum albumin as a function of
time.

[0035] FIG. 16 is a graph showing the stability of PEG-PCL micelles in 4%
bovine serum albumin as a function of time.

[0036] FIG. 17 is a graph showing the stability of PEG-DSPE micelles in 4%
bovine serum albumin as a function of time.

[0037] FIG. 18 is a graph showing the core polarity of PEG-DSPE micelles
for various PEG-DSPE to tocopherol ratios and PEC-DSPE concentrations.

[0038]FIG. 19 is a graph showing the rapamycin loading efficiency by
diffusion-evaporation as a function of rapamycin to amphiphilic polymer
ratio, for ratios of PEG-DSPE:tocopherol at 1:2, 1:1 and no tocopherol.

[0039]FIG. 20 is a schematic of a method of forming PEG-DSPE micelles.

[0040] FIG. 21 is a schematic of a drop wise method of forming polymer
micelles.

[0041] FIG. 22 is a graph showing rapamycin loading efficiency in micelles
as a function of the ratio of rapamycin to amphiphilic polymer.

[0042]FIG. 23 is a graph showing rapamycin release in the presence of
albumin as a function of time in different bovine serum albumin
concentrations.

[0071]FIG. 52 is a chart and a graph showing geldanamycin prodrug
encapsulation in micelles and release over time.

DETAILED DESCRIPTION OF THE INVENTION

[0072] In accordance with the invention, an amphiphilic polymer, a
hydrophobic excipient, and a hydrophobic passenger drug can form a
micelle composition. Methods for making these compositions are also part
of the scope of the invention. In addition, methods of treatment of a
disease or condition utilizing these micelles are part of the scope of
the invention. Micelles incorporated with tocopherol may increase the
drug loading capability of the micelles and also increase the micellar
stability during in vivo conditions. Rapamycin is a drug that
demonstrates impressive activity in the nanomolar range against many
tumor xenograft models, including various solid tumors. In one aspect of
the invention, the low solubility of rapamycin may be overcome by
incorporating rapamycin into micelle compositions for delivery to target
tumor sites.

1.0 Micelles

[0073] Nonionic surfactants, such as Cremophor EL and Tween 80, may be
used for intravenous administration of cancer treatments. As shown in
FIG. 1, micelles are supermolecular structures having a core-shell form.
Micelle formation is entropy driven. See FIG. 2. Water molecules are
excluded into a bulk phase. ΔG0mic=RT ln(CMC) informs the
formation of micelles. When above critical micelle concentration (CMC),
amphiphilic unimers aggregate into structured micelles. Polymeric
micelles are spherical and may have nanoscopic dimensions typically in
the 20-100 nm range. This is advantageous as circulating particles should
be less than about 200 nm to avoid filtering by the interendothelial cell
slits at the spleen. Polymeric micelles have been shown to circulate in
the blood for prolonged periods and capable of targeted delivery of
poorly water-soluble compounds. Upon disassociation, micelle unimers are
typically <50,000 g/mol, permitting elimination by the kidneys.
Ideally, this allows prolonged circulation with no buildup of micelle
components in the liver that could lead to storage diseases.

1.1 Amphiphilic Polymers

[0074] Polymers that can encapsulate poorly-water soluble drugs include:
pegylated phospholipids and pegylated poly-ε-caprolactone. These
polymers exhibit high biocompatibility and solubilization capacity for a
broad range of compounds. Coexcipients, such as α-tocopherol, can
substantially increase the drug loading capacity of micelles formed from
these polymers and allow solubilization of potential drug candidates
previously thought incompatible or poorly solubilized by existing
polymeric carriers.

[0075] Amphiphilic polymers are typically composed of a hydrophilic
domain, e.g. polyethylene glycol (PEG), and a hydrophobic domain such as
poly(propylene glycol), poly(L-amino acid), poly(ester), and
phospholipids. These polymers can assemble into polymeric micelles,
highly ordered supramolecular core-shell structures having a hydrophobic
interior capable of encapsulating small hydrophobic compounds and a
hydrophilic exterior. As shown in FIG. 3, the micelle core has low
polarity and is a hydrophobic environment. There is a high core capacity
for hydrophobic compounds. There can be up to about 4:1 drug:polymer
loading. The micelle core can increase in solubility of up to about
30,000 times. The micelle corona is hydrophilic.

[0076] Polymeric micelles have been shown to circulate in the blood for
prolonged periods and are capable of targeted delivery of poorly
water-soluble compounds. Example 1 illustrates that drugs such as
doxorubicin and paclitaxel can be encapsulated in micelles and targeted
to tumors.

[0077] The key benefits of micelle compositions include ease of storage
and delivery; compositions may be lyophilized and reconstituted before
intravenous administration. This lowers the risk of drugs precipitating
and causing an embolism. Micelle compositions are capable of long blood
circulation, low mononuclear phagocyte uptake, and low levels of renal
excretion. Also, micelle compositions have enhanced permeability and
retention (EPR) to increase the likelihood of the chemotherapeutics
reaching tumors. As shown in FIG. 4, tumors have high vascular density as
well as defective vasculature so high extravasation occurs. There may be
impaired lymphatic clearance. The endocytosis and subsequent drug release
increases the effect of the chemotherapeutics on the tumor.

[0078] Initial studies have focused on PEG-DSPE (FIG. 5) and the block
co-polymers and PEG-PCL (FIG. 6) for drug solubilization. PEG-DSPE may be
a safe and effective micelle carrier for both chemotherapeutic agents.
PEG-PCL is biodegradable and may have biocompatibility.

[0079] The principal difference between neutral PEG-DSPE and negatively
charged PEG-DSPE membranes is the electrostatic force between the two
charged membranes. Membrane charges affect the adsorption of acidic and
basic proteins on charged and neutral membranes. This may alter the
interactions of various proteins with the bilayers. These differences may
be responsible for the differences in opsonization and phagocytosis of
neutral versus charged liposomes. The phosphate group at the hydrophobic
head of PEG-DSPE may affect the tightness of the PEG-DSPE's at the
core-water interface due to electrostatic repulsion. Also, this charged
nature may influence protein interaction with the hydrophobic core should
the protein penetrate the PEG corona. Tocopherol (FIG. 7) has been shown
to interpolate between the phospholipid head groups and the
ring-structure at the head of the tocopherol may prevent further protein
penetration and interaction. See FIG. 8. Also, the tocopherol head group
and hydroxyl group have been shown to act as an antioxidant and may
prevent protein disruption of the phospholipid layer. PEG-b-PCL may be
biocompatible and biodegradable. PEG-b-PCL may have a low critical
micelle concentration (CMC). A PEG:PCL ratio of about 5:6 may have a CMC
of under about 0.5 μM. PEG-PCL may have a rigid core structure and be
stable in the presence of albumin.

[0080] The choice of polymeric micelle compositions can be highly
dependent on the structural relationship between the target drug compound
and the hydrophobic core of the carrier. The use of tocopherol may also
modify the core properties of the micelles so as to induce higher loading
of drugs which are otherwise poorly soluble in the micelle of study.

2.0 Passenger Compounds

[0081] In accordance with the invention, drugs can be passenger compounds
in polymer carriers. Such drugs include: rapamycin (FIG. 9), geldanamycin
(FIG. 10), and paclitaxel (FIG. 11). These drugs are potent small
molecule chemotherapeutic agents with unique targets of action. Studies
of these compounds and the development of clinical products have been
hampered by their extremely low water solubilities, for example,
rapamycin ˜2.6 μg/ml and geldanamycin ˜1.5 μg/ml. Using
combinations of the above polymeric compounds and integrating tocopherol
into the micelle structure, stable micelle solutions of these compounds
were achieved incorporating up to about 5 mg/ml of rapamycin, a 1900-fold
increase in solubility, and up to about 500 μg/ml of geldanamycin, a
300-fold increase. In addition, using prodrugs of geldanamycin or
paclitaxel significantly increase solubilities.

[0082] The promise of these compounds as chemotherapeutics merits their
further evaluation with in vitro and in vivo tumor models. The successful
formulation of these compounds using the phospholipids and
poly-caprolactone/tocopherol systems merits investigating their
application to other hard-to-solubilize drug compounds.

[0083] The choice of polymeric micelle carrier can be highly dependent on
the structural relationship between the target passenger drug compound
and the hydrophobic core of the carrier. Less than 3% (w/w) paclitaxel
may be loaded into PEG-PCL micelles. However, PEG-poly(D,L-lactide)
micelles have a loading capacity>20% (w/w). Therefore, conditions of
polymeric micelle carriers must be optimized for loading a desired
passenger compound.

2.1 Rapamycin

[0084] The formulation of these compounds, especially rapamycin, for
intravenous delivery without the use of co-solvents, e.g., ethanol or
polyethylene glycol, permits them for therapeutic usage. The use of
micelle carriers allows delivery of therapeutic dosages of this drug
without chemical modification. In addition, micelle delivery allows
targeted treatment to tumors through the EPR effect, reducing the
likelihood of immunosuppression, a side-effect of free rapamycin and its
water soluble derivatives.

[0086] A novel mechanism may have rapamycin binding to FK506-12, in which
rapamycin inhibits mTOR growth regulators, prevents G1 to S phase
transition, and inhibits NF-kB and enhances apoptosis.

[0087] Unfortunately, rapamycin is practically insoluble in water
(˜2.6 μg/ml) and has no ionizable groups. The targeted delivery
and retention of rapamycin to tumor sites, using the EPR effect, may
substantially increase its potency. In addition, targeted delivery may
attenuate the side effects of rapamycin treatment including
immunosuppression. The retention of rapamycin's native hydrophobic nature
may be important in neurological applications where modification (to
increase water solubility) may hinder crossing of the blood brain
barrier.

[0088] Using polymeric micelles, rapamycin can be solubilized in large
quantities--well within the range required for clinical feasibility.
Rapamycin has been solubilized using PEG-PCL and PEG-DSPE micelles with
the addition of tocopherol. Results are summarized in Example 2.

2.2 Geldanamycin

[0089] Geldanamycin (FIG. 10) is a member of the new class of compounds
known as heat shock protein inhibitors, having both anti-tumor and
neurological disease applications. The mode of action is inhibiting heat
shock protein 90 (Hsp90), strongly binding to Hsp90 (Kd=1.2 μM),
and preventing interaction with downstream components. This in turn leads
to ubiquitination of a broad range of oncogenic client proteins and their
subsequent degradation.

[0090] Hsp90 inhibitors may be useful in drug resistant cancers by
inducing different pathways, such as in rapamycin resistant tumors.
Despite the promise of Hsp90 inhibitors, such as geldanamycin, the
clinical progression of these therapies has been slow due to the lack of
a suitable formulation. Radicicol, an Hsp90 inhibitor, is also unstable
in vivo. Geldanamycin has extremely poor water solubility, and is
hepatotoxic in vivo (MTD dog<100 mg/m2). Geldanamycin prodrugs
such as 17-AAG have slightly better solubility and lower hepatoxicity
(MTD dog 500 mg/m2), but are still difficult to formulate, requiring
toxic excipients such as Cremaphor, Tween 80, and DMSO. Water soluble
prodrugs of geldanamycin, such as 17DMAG (MTD dog 8 mg/m2), may
avoid these formulation problems, but the wide biodistribution and
increased toxicity of these prodrugs may present additional difficulties.

[0091] For clinical formulations, a solubility of at least about 1 mg/ml
is desirable. Phase I results found GI toxicity to be dose limiting for
17-AGG, with a suggested Phase II dosing of 40 mg/m2. Preclinical
trials found severe hepatotoxicity to be dose limiting for the parent
compound, geldanamycin (4 mg/kg).

[0092] By targeting multiple oncogenic proteins, geldanamycin promises
efficacy against a broad range of tumors and increases the chances of
overcoming drug resistance. In addition, the inhibition of Hsp90 leads to
an up-regulation of Hsp70, which reduces the formation of abnormal tau
species, the primary component of plaque deposits in Alzheimer's and
Parkinson's disease.

[0093] Because of the extremely low water solubility of geldanamycin,
˜1.5 μg/ml, formulations have used various soluble analogs such
as 17-AAG. As with rapamycin, the targeted delivery of geldanamycin to
tumor sites and the EPR effect are expected to substantially increase its
potency. In addition, prolonged circulation time and reduced liver
retention should dramatically reduce hepatotoxicity. Finally, the
possible advancement of geldanamycin as a treatment in neurological
diseases will require the highly hydrophobic nature of the parent
compound, which is attenuated in soluble analogues, in order to cross the
blood-brain barrier.

2.3 Paclitaxel

[0094] Paclitaxel is another hydrophobic compound with applications
including the treatment of cancer. Paclitaxel belongs to a group of
medicines called antineoplastics, which inhibit cellular growth. The
inhibition is accomplished by disrupting microtubule function by binding
to the beta subunit of tubulin. The disrupted microtubule looses the
ability to disassemble, a necessary function, for example, in chromosomal
migration during cell replication. Additionally, research has indicated
that paclitaxel induces apoptosis, programmed cell death, by binding to
an apoptosis stopping protein called Bcl-2 and stopping its function.

3.0 Excipients

[0095] Multi-component excipients may be used in drug formulations, where
a poorly water soluble component solubilizes the drug compound in
addition with a second excipient or co-solvent. The solubilization
capacity and stability of polymeric micelles may be enhanced by the
inclusion of a co-excipient highly compatible with both the hydrophobic
micelle core formed by the micelle unimers and the loaded drug.

[0096] Multi-component excipients may be used in drug formulations, where
a poorly water soluble component solubilizes the drug compound in
addition with a second excipient or co-solvent, e.g., risperidone oral
formulation containing benzoic acid, tartaric acid, and sodium hydroxide.
The solubilization capacity and stability of polymeric micelle
compositions may be enhanced by the inclusion of a co-excipient highly
compatible with both the hydrophobic micelle core formed by the micelle
unimers and the loaded drug.

[0097] Excipients may have a high Po/w, preferably greater than about 3.5,
and a low molecular weight, preferably less than 1000 Da. Excipients may
improve biocompatibility and may improve drug-carrier compatibility or
increase the drug loading and release time from the carrier.

3.1 Tocopherol

[0098] The ring and alkyl chain structure of α-tocopherol (FIG. 7),
the most common isomer tocopherol, is a feature common to many
poorly-soluble drugs, hence tocopherol's long history as an excipient for
many difficult to formulate drugs. Tocopherol may also be a modifying
agent to micelle structures. Drug loading capacities of PEG-DSPE and
PEG-PCL micelles are significantly enhanced by the addition of
tocopherol. See Example 2.

[0099] The inclusion of tocopherol may also enhance the stability of
micelles. For example, PEG-DSPE micelles can be formed with up to about 4
mg/ml of rapamycin, however, the micelles quickly "crash" causing the
drug to come out of solution (typically <2 hours). The same micelles
with the incorporation of tocopherol are stable for at least several
days. See Example 3 and 6. The critical micelle concentration increases
with the incorporation of tocopherol into the micelle compositions,
thereby increasing the kinetic stability of the micelle composition. See
FIG. 13.

[0100] The phytol chain of tocopherol interpolates between phospholipid
acyl chains. When a phase has a tocopherol:phospholipids ratio greater
than 0.2:1 then the phase is a tocopherol-rich phase. FIG. 8 shows the
tocopherol incorporation between PEG-DSPE chains. Tocopherol
incorporation results in the formation of separate tocopherol phase. The
mobility of mixed acyl and phytol chains are decreased after tocopherol
incorporation. There is a kinetic contribution of polymers to micelle
composition stability. The micelle unimer exchange rate is slow with a
highly viscous, or rigid, core. A reduced core viscosity, or rigidity may
increase diffusion rate of the passenger drug. FIG. 13 shows the core
rigidity data. As the tocopherol to PEG-DSPE ratio increases, the core
rigidity generally decreases. An increase in the hydrophobic core size,
influenced by the addition of tocopherol, may modulate the drug diffusion
rate. The increased core size causes the drug to travel a further
distance, but the less viscous core allows the drug to travel faster. If
there is not optimized interaction between the tocopherol and the drug,
then diffusion may be slowed. Tocopherol and drug incorporation into a
micelle composition may affect the size of the micelle and thus affect
extravasation at the tumor site. See Example 9 and FIG. 14. As shown in
FIG. 15, PEG-DSPE micelles are stable in phosphate buffered saline
solution, but are unstable in 4% bovine serum albumin which approximates
in vivo conditions. FIG. 16 shows PEG-PCL is stable in a 4% albumin
serum. As shown in FIG. 17, PEG-DSPE micelle compositions with
incorporated tocopherol (at about 2:1 ratio of tocopherol:PEG-DSPE) stay
about 60% solubilized in 4% bovine serum albumin for about 25 hours. See
Example 6.

[0101] As seen in Example 3, the critical micelle concentration (CMC)
increases with the incorporation of tocopherol into the micelle
composition. Micelle compositions are formed between 10-6 and 10 M
PEG-DSPE. The PEG-DSPE:tocopherol ratio and the effect on the CMC are
described in Example 3.

[0102] As shown in FIG. 18, the core polarity of a micelle composition
with incorporated tocopherol also changes with the proportion of
tocopherol. The core polarity decreases with the greater incorporation of
tocopherol.

[0103] Rapamycin and tocopherol are both very hydrophobic and have similar
structural components. Both have ring structures and long alkyl chains.
Both may increase stability of drug incorporation within micelle
compositions.

[0104] As shown in FIG. 19, rapamycin loading efficiency increases with
the incorporation of tocopherol at all rapamycin to PEG-DSPE ratios. The
most effective tocopherol to PEG-DSPE ratio is about 2 and about 4, both
ratios leading to a loading efficiency around 25%.

4.0 Result of Micelle and Drug Incorporation

[0105] Tocopherol may have effects on the structure and properties of
PEG-DSPE and PEG-PCL micelles. Briefly, PEG-DSPE2000 micelles were
prepared according to the solvent film method of Lukyanov et al. (as
summarized in FIG. 20), wherein, phospholipids, additives, and drug were
dissolved in an organic solvent, evaporated to produce a dry film, and
micelles were formed by the addition of water. Micelles were then
filtered and/or centrifuged to remove unincorporated drug aggregates and
drug incorporation verified by Size Exclusion Chromatography (SEC).
PEG-DSPE2000 used in this process may have a concentration between
about 1 mM and about 20 mM, preferably between about 1.5 mM and about 10
mM, and most preferably about 5 mM. Tocopherol used in this process may
have a concentration between about 1 mM and about 20 mM, preferably
between about 2 mM and about 15 mM, and more preferably about 10 mM. The
phospholipids, additives, and drug dissolved in an organic solvent may be
spun at between about 50 rpm and about 200 rpm, preferably between about
70 rpm and about 150 rpm, and most preferably about 100 rpm. Solvent may
be removed by vacuum at between about 1 and about 500 μbar, preferably
between about 5 and about 200 μbar, and most preferably between about
10 and about 100 μbar.

[0106] As described in FIG. 21, PEG-PCL micelles were also prepared by the
drip-wise addition of drug and PEG-PCL dissolved in a miscible solvent,
acetone, to vigorously stirred water, followed by removal of the solvent
by N2 purge, and 0.2-μm filtration and/or centrifugation. The
final solvent to water ratio is between about 0.1 and about 5, preferably
between about 0.5 and about 4, and more preferably about 2. The micelle
solution should be delivered at a rate of between about 2 s/drop and
about 60 s/drop, preferably between about 5 s/drop and about 30 s/drop,
and more preferably between about 10 s/drop and about 20 s/drop.

[0107] As shown in FIG. 22, rapamycin loading by the solvent film method
had a loading efficiency of between about 30% and about 50%, preferably
between about 32% and about 47% and more preferably about 40% at a
rapamycin to PEG-DSPE ratio of about 2:1. The weight % of rapamycin at
the ratio of 2:1 is between about 10% and about 40%, preferably between
about 15% and about 30%, and more preferably about 20%.

[0108] Rapamycin, as shown in FIG. 23, stays solubilized for a longer
period of time when loaded into a micelle composition compared to a free
drug under in vivo conditions. As shown in FIG. 24, PEG-DSPE is unstable
in the presence of human serum albumin.

4.1 Micelle Composition Properties with the Incorporation of Tocopherol

[0109] Tocopherol alters the core structure of PEG-DSPE as expected based
on studies with unpeglylated DSPE micelles. As shown in Example 3, the
addition of up to a 2:1 molar ratio of tocopherol to PEG-DSPE2000
micelles increased the critical micelle concentration (CMC) from 2.1
μM to 28 μM, but this CMC range is still indicative of a very
stable micelle. Likewise, PEG-PCL micelles retained very low CMC's at 10
and 20:1 ratios of tocopherols to PEG-PCL unimers. As shown in FIG. 18,
tocopherol incorporation decreases core polarity and may increase the
loading of lipophilic molecules.

[0110] The addition of tocopherol did not increase the size of micelles
formed with PEG-DSPE. This may be due to the incorporation of tocopherol
into the alkyl chains and minimal swelling of the hydrophobic core
(Example 6). However, the PEG-PCL micelles increased in size with the
addition of tocopherol. As shown in FIG. 25, tocopherol incorporation
does not affect the size of the micelle composition significantly. As
shown in FIG. 14, the increasing aggregate number of incorporation also
reflects an increasing size of the core. At a tocopherol to lipid ratio
of 0.5, the change in aggregate number became statistically significant.
This may in part be due to the greater loading of tocopherol into the
PEG-PCL micelles.

4.2 Micelle Properties with Incorporation of Tocopherol and Passenger
Drugs

[0111] Rapamycin or geldanamycin may be loaded into PEG-DPSE and PEG-PCL
micelles with varying amounts of tocopherol. See Example 1. As shown in
FIG. 26, rapamycin may be loaded into PEG-DSPE micelles. The loading of
rapamycin may be increased by between about 2 and about 7 fold,
preferably between about 4 and about 6 fold, and more preferably over
3-fold by the addition of tocopherol to PEG-DSPE and PEG-PCL micelles. In
addition, in the absence of tocopherol, precipitation may be observed
after 1-4 hours; this indicated that tocopherol may increase the
stability of drug loaded PEG-DSPE micelles. See Example 10. Tocopherol
increased the loading of geldanamycin into PEG-DSPE micelles by between
about 1 and about 4 fold, preferably between about 1 and about 3 fold,
and more preferably about 2 fold and the loading into PEG-PCL micelles by
between about 7 and about 15 fold, preferably between about 8 and about
12 fold, and more preferably about 10 fold.

[0112] The human body is like a perfect sink. As shown in FIG. 27, Crank's
solution for Fickian diffusion informs the diffusion of the drug from the
micelle composition.

[0113] The benefits of tocopherol were most dramatic in the case of
geldanamycin and PEG-PCL. Without the addition of tocopherol, PEG-PCL may
be ineffective as a solubilization agent. The maximal loading
concentration of between about 0.2 and about 0.8 mg/ml, preferably
between about 0.4 and about 0.6 mg/ml, and more preferably 0.5 mg/ml may
be achieved with the 1:20 PEG-PCL:tocopherol. See Example 11 and 12.
Further optimization of the carrier and additives may be required. Also,
the EPR effect of micelle composition formulations may reduce the dosage
requirements for chemotherapy.

[0114] As shown in FIG. 28, tocopherol increases the time over which
rapamycin is released in a phosphate buffered solution, but not
significantly so. In FIG. 29, tocopherol is shown as having a significant
effect on the increased time over which rapamycin is released in a 4%
bovine albumin solution.

[0116] Early results demonstrate the potential these polymers have as
carriers for chemotherapeutic compounds. Results with tocopherol
demonstrate that structurally similar additives can substantially
increase drug loading capacity.

4.3 Dosage for Micelle Administration

[0117] The dose of rapamycin through micelle a micelle delivery system can
be similar to doses used in clinical trials for rapamycin analogues:
CCI-779, RAD-001, and AP-23573. The doses for CCI-779 is about 7.5 to 220
mg/m2/week i.v., about 0.75 to 20 mg/m2/day i.v. for about 5 days every 2
to 3 weeks, about 25 to 100 mg/day p.o. for about 5 days every 2 weeks.
For RAD-001, about 5 to 60 mg/week p.o. For AP-23573, about 6.0 to 100
mg/week i.v., about 3 to 30 mg/day i.v. for about 5 days every 2 weeks.
These doses should be easily attained by PEG-b-PCL micelles, given
solubilization of rapamycin at about 1 to 4 mg/ml. The content of
rapamycin in PEG-b-PCL micelles is about 10 to 20% wgt drug/wgt polymer.
PEG-b-PCL micelles can reach at least about 40 mg/ml.

[0118] The dose of geldanamycin prodrugs can be about 100 to 1000
mg/m2 at about 1 to 7 mg/ml, preferably about 200 to 700 at about 2
to 6 mg/ml, even more preferably at about 100 ml at about 4.0 mg/ml.

4.4 Geldanamycin Prodrugs Loading into Micelles

[0119] As shown in FIG. 42, geldanamycin loads poorly into PEG-b-PCL
micelles and into PEG-DSPE micelles due to not being lipophilic enough.
As shown in FIGS. 43 and 44, fatty acid (ester) prodrugs of geldanamycin
may increase lipophilicity. As shown in FIG. 14, increasing the log Po/w
increases the loading percentage by weight of a geldanamycin prodrug. See
Example 18.

[0120] In the design of a nanocarrier, a major concern must be
drug-carrier interaction. Initial studies found that geldanamycin may not
be sufficiently encapsulated by nanocarriers such as PEGylated
phospholipids and PEG-b-polycaprolactone (PEG-PCL) micelles.
Encapsulation of Hsp90 inhibitors may be dependent on hydrophobicity of
the drug molecule. The octanol-water partition coefficient of
geldanamycin was determined by microemulsion electrokinetic
chromatography. As a comparison, rapamycin, which was loaded to high
levels (>10% w/w) in PEG-PCL micelles, has a log Po/w of 3.77, as
determined by MEEKC.

[0121] Several prodrugs were synthesized by DMAP/DCC chemistry, as shown
in FIG. 44. As shown in FIGS. 45 and 46, extending the fatty acid chain
length increases the hydrophobicity of the resulting molecule, resulting
in a higher value log Po/w. The addition of a bromine adjacent to the
carbonyl of the ester acts as an electron withdrawing group,
destabilizing the ester bond. However, bromine (Br) is extremely
hydrophobic and increases the molecule's overall log Po/w coefficient.
The addition of the Br may also increase loading into the nanocarrier,
but may reduce the accessibility of hydronium and hydroxide ions to the
ester bond, decreasing the hydrolysis rate of the encapsulated esters. In
turn, slow hydrolysis may prolong the drug release rate if the prodrug
partitions into the micelle core significantly better than the parent
drug. A highly partitioned drug, with a stable ester bond, may be
realized if the Br is replaced with a hydrophobic group which is not
electron withdrawing, such as an isopropyl group, shown in FIG. 47.

[0122] As shown in Table 1, geldanamycin prodrugs are highly hydrophobic,
as evidenced by the high log Po/w values. Unmodified geldanamycin has a
log Po/w value of about 2.77, which is not hydrophobic enough to be
encapsulated by PEG-b-PCL. Effective encapsulation by PEG-b-PCL may occur
when the carrier has a hydrophobicity of about 3.5 or higher. The
compound 17-aminoethyl-hexonate-17-demethoxygeldanamycin has a log Po/w
of about 3.87, which is enough to allow the molecule to be substantially
encapsulated into a micelle, such as PEG-b-PCL. The compound
17-aminoethyl-bromohexonate-17-demethoxygeldanamycin is a very
hydrophobic molecule with a log Po/w at about 4.49 and should encapsulate
into a micelle, such as PEG-b-PCL.

[0124] FIG. 45 shows an extension of a fatty acid chain. In the first
step, the addition of ethanol amine to geldanamycin (shown as 1 in FIG.
45) may be accomplished by dissolving geldanamycin in chloroform with
about 10 equivalents of ethanol amine for between about 1 and about 4
hours. The reaction is monitored by thin layer chromatography (TLC) until
complete. The organic layer is washed with sodium bicarbonate
(NaHCO3) and then brine. The organic layer is then dried over sodium
sulfate (NaSO4) and then the solvent is removed by rotary
evaporation.

[0125] In the second step of FIG. 45, a fatty acid chain is added to the
geldanamycin prodrug structure shown as 2, by a DMAP/DCC reaction. A
fatty acid is added with a hydrophobic entity (such as Br or H) adjacent
to the carbonyl of the ester. In the second step, the geldanamycin
prodrug from 2 is suspended in about 10 ml of dichloromethane having
about 1.5 equivalents of the fatty acid, about 3 equivalents of DCC and
about 1 equivalent of DMAP. The reaction is monitored by TLC for between
about 2 and about 6 hours until completion. The solution is chilled and
filtered. The solution is then purified by flash chromatography on silica
loaded with about 1:9 methanol:chloroform. The solution is then
rotovapped to obtain the product.

[0126] FIG. 46 shows the process for formulating
17-amino-hexyldecyl-17-demethoxygeldanamycin. FIG. 46 shows a different
first step from FIG. 45, but the same second step. In the first step, the
addition of NH2(CH2)15CH3 amine to geldanamycin
(shown as 1 in FIG. 45) may be accomplished by dissolving geldanamycin in
chloroform with about 5 equivalents of NH2(CH2)15CH3
for between about 1 and about 4 hours. The reaction is monitored by thin
layer chromatography (TLC) until complete. The organic layer is washed
with sodium bicarbonate (NaHCO3) and then brine. The organic layer
is then dried over sodium sulfate (NaSO4). The solution is then
purified by flash chromatography on silica and eluted with about 1:9
methanol:chloroform. The solution is then rotovapped to obtain the
product.

[0127]FIG. 47 shows the process for formulating
17-hydroxyethylamino-(1-isopropyl-palmitate)-17-demethoxygeldanamycin.
This is made by suspending diethyl malonate in about 1 equivalent of
NaOCH2CH3 in ethanol and refluxing for about 1 hour. Then about
0.95 equivalents of 2-bromo-isopropane is added dropwise and refluxed for
about 4 hours. Twice the volume of cold water is added to the solution.
The product is extracted three times by ether and then vacuum distilled.
The isopropylmalonate diester is mixed with about 1 equivalent of
NaOCH2CH3 in ethanol and refluxed for about 1 hour. Then about
0.95 equivalents of 1-bromotetradecdane is added and the solution is
refluxed for about 4 hours or until complete by TLC. About twice the
volume of cold water may be added to the solution. The product may be
extracted three times by ether and then vacuum distilled.

[0128] Then 2-isopropyl-2-tetradecdane-malonatediester may be dissolved in
about 1:1 KOH:water and refluxed for about 8 hours. Then water is added
until the solids are gone. The aqueous layer is extracted. Concentrated
hydrochloric acid is added until there are no more solids. The solution
is extracted with ether three times, and reduced in a vacuum. The product
is then heated to about 180 degrees C. for about 3 hours and then vacuum
distilled. This results in the fatty acid with isopropyl shown as 3 in
FIG. 2. Then the geldanamycin prodrug in 2 in FIG. 1a is mixed with 3 in
FIG. 2. The geldanamycin prodrug is mixed with about 1.5 equivalents of
the fatty acid containing isopropyl with about 3 equivalents of DCC and
about 1 equivalent of DMAP in about 10 ml of dichloromethane for between
about 2 and about 6 hours. The solution is chilled and filtered. The
solution is then purified by flash chromatography on silica loaded with
about 1:9 methanol:chloroform. The solution is then rotovapped to obtain
geldanamycin-C17-aminoethyl-2-isopropylhexadecanoate.

[0129]FIG. 48 shows the process for formulating
geldanamycin-C17-aminoethylonate-Phe-Leu-Phe-amine. The hydrophobic
peptide is added to the geldanamycin prodrug shown as 2 in FIG. 45. Three
equivalents of DCC and 1 equivalent of DMAP are added along with about 10
ml of dichloromethane. The reaction time may be between about 2 and about
6 hours. The solution is chilled and filtered. The solution is then
purified by flash chromatography on silica loaded with about 1:9
methanol:chloroform and then rotovapped. The resulting product is mixed
with about 2:8 piperidine:DMF and reacted for between about 1 and about 2
hours. The solution is then purified by flash chromatography on silica
loaded with about 1:9 methanol:chloroform. The solution is then
rotovapped to obtain geldanamycin-C17-aminoethylonate-Phe-Leu-Phe-amine.

[0130]FIG. 49 shows the process for formulating
geldanamycin-C17-aminoethylidene-palmitohydrazide. Fmoc-ethanolamine may
be converted to the aldehyde using about 1 equivalent of Dess-Martin in
DCM. After about 20 minutes, the reactions may be diluted with about 1
volume of saturated sodium bicarbonate and about 7 equivalents of
saturated sodium thiosulfate. The reaction may be stirred for about 20
minutes and extracted about 3 times with substantially equal volumes of
diethyl ether. The organic then may be washed with about 1M HCl and
H2O, dried over sodium sulfate, and the solvent removed by rotary
evaporation. The product was purified by flash chromatography on silica
and eluted with about 99:1 EtOac:TEA. The Fmoc-ethylaldehyde may be mixed
with about 1 equivalent of palmitic acid hydrazide and refluxed overnight
in EtOH.

[0131] The Fmoc-hydrazide product may be purified by flash chromatography
on silica and eluted with about 89:10:1 chloroform:MeOH:TEA. The
Fmoc-hydrazide may be deprotected in about 2:2:98 DBU:piperidine:DMF
overnight at room temperature. The product
(E)-N'-(2-aminoethylidene)palmitohydrazide may be filtered and purified
by flash chromatography with about 89:10:1 chloroform:MeOH:TEA. The
hydrazide was then conjugated to geldanamycin in DMF by nucleophilic
attack at the C17-methoxy. The product,
17-(2-aminoethylidene)palmitohydrazide-17-geldanamycin, was purified by
flash chromatograpy on silica eluted with 1:9 MeOH:chloroform.

[0132]FIG. 50 shows the process for formulating PEO-b-PEGA. PEO-b-PBLA is
aminolysed with HOOC(CH2)5NH2 in DMF and
2-hydroxypyridines, thus incorporating a hydroxyl moiety. The product is
then conjugated to 17-hydroxyethyl-amino-17-geldanamycin using DCC/DMAP
chemistry in DCM. The product may be purified by cold filtering and ether
precipitation.

[0133] Increasing the hydrophobicity of geldanamycin may increase the
nanoencapsulation of the compound. Prodrugs of geldanamycin at the 17
carbon have been shown to have less impact on bioactivity of geldanamycin
than other positions; however, derivatization often leads to a decrease
in activity, especially large groups (Sasaki et al, U.S. Pat. No.
4,261,989 (1981)).

[0134] Sasaki showed that the
β-hydroxyethylamino-17-demethoxygeldanamycin prodrug had minimal
impact on bioactivity in vitro. This prodrug provides a hydroxyl group
allowing esterification. Ester prodrugs may hydrolyze into the active
form of the parent compound

[0135] Modifications to geldanamycin are not limited to those listed
above. Instead of fatty acids, hydrophobic peptide sequences could be
used, and, for example, attached via the terminal C-group using an ester
bond. For example, a sequence of phenylalanines and leucines may be used.
The sequence may alternate between amino acids to prevent the formation
of extensive secondary structures. A representative prodrug,
C17-amino-ester-Phe-Leu-Phe is shown in FIG. 48. Amino acids may be
assembled using standard solid phase peptide chemistry, e.g. Fmoc
protected amino acids, with HATU/HOAt activated coupling. The resulting
N-protected peptide may be conjugated using by DMAP/DCC chemistry as in
FIG. 47. After conjugation, the terminal amino acid Fmoc protecting group
may be removed.

[0136] Other groups besides esters may be used for attachment of
hydrophobic groups, for example hydrazone linkers may be used that have
the advantage of stability at neutral pH and enhanced hydrolysis at
acidic conditions. Tumors may present an acidic environment that may
enhance release of the drug, while the drug may be stable in the
nanocarrier JM plasma, reducing non-specific release and resulting
toxicity. An example of one linker is shown in FIG. 44.

[0137] The Hsp90 drug may also be linked using other bonds such as acetyl
and disulfide bonds, cleavable peptide bonds (eg. Ala-Val), or a
combination of these linkers. For example, a tumor selectively-cleaved
linker (e.g. Ala-Val peptide) may be attached via the C-terminus to a
fatty acid or hydrophobic peptide. The N-terminus may be linked directly
to the Hsp90 inhibitor (e.g. via the C17 carbon of geldanamycin) or via a
spacer linker such as an aminoethanol or aminohexanol. The N-terminus may
also be linked via another cleavable linker. The resulting compound may
show reduced non-specific toxicity after nanocarrier release due to the
bulky Ala-Val-(drug linker) groups reducing drug affinity to Hsp90. After
tumor specific cleavage of the Ala-Val, the resulting compound may show
sufficient Hsp90 binding for inhibition.

[0138] The Hsp90 inhibitor may also be linked to the nanocarrier. If
linked reversibly, the drug may release from the nanocarrier and become
bioactive. If linked irreversibly or reversibly, the presence of the
bound drug may increase the partitioning of free drug into the micelle.
An example is shown in FIG. 45 using PEO-β-PEGA as the carrier.

[0139] These modified Hsp90 inhibitors may show sustained release from the
carrier. The release kinetics of several of these carriers are shown in
Table 2. Drugs were loaded into 0.5 mM PEG-b-PCL (5000:10000 Da) micelles
to achieve a 25% wt loading (or 1.9 mg/ml solution). These data were
obtained by measuring release from 10000MWCO dialysis cassettes into pH
7.4 phosphate buffer under perfect sink conditions at 37° C. Drug
diffusion was calculated as described in Forrest and Kwon, 2005 (Journal
of Controlled Release).

[0140] PEG-PCL micelles are prepared by the drop-wise addition of
geldanamycin prodrug and PEG-PCL dissolved in a miscible solvent,
acetone, to vigorously stirred water, followed by removal of the solvent
by N2 purge, and 0.2-μm filtration. Alternatively, the solution
may be centrifuged to remove unincorporated and aggregated drug. The
final solvent to water ratio is between about 0.1 and about 5, preferably
between about 0.5 and about 4, and more preferably about 2. The micelle
solution should be delivered at a rate of between about 2 s/drop and
about 60 s/drop, preferably between about 5 s/drop and about 30 s/drop,
and more preferably between about 10 s/drop and about 20 s/drop.

[0141]FIG. 51 is a graph showing the loading of timed release of
geldanamycin prodrugs, with dodeconate, bromododeconate, and
aminohexyldecyl, C16-amino-geldanamycin, and
C16-bromo-ester-geldanamycin. PEG-PCL micelles including
C16-ester-geldanamycin may carry about 1.1 mg/ml of the drug and may be
an about 13 wt % carrier. PEG-PCL micelles including
C16-amino-geldanamycin may carry about 1.1 mg/ml of the drug and be an
about 14 wt % carrier. PEG-PCL micelles including
C16-bromo-ester-geldanamycin may carry about 1.1 mg/ml of the drug and be
an about 14 wt % carrier.

[0142] Cytotoxicities of the drugs to the MDA-MB-468 breast cancer cell
line (ATCC) were determined. Cells are plated at a density of 3000
cells/well into 96 well plates (100 μl/well DMEM medium). After 24
hours, drugs were added dissolved in 1% DMSO. Cells were incubated with
drugs for 4 days and toxicity determined using the MTS cytotoxicity assay
according to manufacturer's directions (Promega, Madison, Wis.).

[0143] Because hydrolysis of the linkers may be slow, the toxicity may be
enhanced upon exposure times greater than 4 days.

[0144] A Cremephor® and solvent free formulation of paclitaxel was
prepared using amphiphilic block co-polymer micelles of poly(ethylene
glycol)-b-poly(ε-caprolactone) (PEG-PCL). The poor loading of
paclitaxel in micelles of PEG-PCL (<1% w/w) was overcome by forming
hydrolysable fatty acid prodrugs of paclitaxel. Paclitaxel prodrugs had
solubilities in excess of 5 mg/ml in PEG-PCL micelles. Drug loaded
PEG-PCL micelles were prepared by a co-solvent extraction technique.
Resulting PEG-PCL micelles contained 17-22% w/w prodrug and were less
than 50 nm in diameter. PEG-PCL micelles released paclitaxel prodrugs
over several days, t1/2>3 d.

[0145] 5.0 Different Aspects of the Invention

[0146] In summary, a micelle composition may comprise an amphiphilic
polymer, a hydrophobic excipient, and a hydrophobic passenger drug. The
amphiphilic polymer may be a pegylated phospholipids, such as PEG-DSPE,
or a block copolymer, such as PEG-b-PCL and PEG-b-amino acids. The
hydrophobic excipient may have a log Po/w greater than about 3.5 and a
molecular weight less than about 1000 Da. The hydrophobic excipient may
be Vitiamin E, which has many isomers, including: alpha-tocopherol,
beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol,
beta-tocotrienol, gamma-tocotrienol, delta-tocotrinol. The hydrophobic
passenger drug may be geldanamycin, geldanamycin prodrug, rapamycin,
paclitaxel, or a paclitaxel prodrug.

[0147] A micelle composition may be an amphiphilic polymer and a
hydrophobic passenger drug may be utilized for a micelle. The hydrophobic
passenger drug may be geldanamycin, geldanamycin prodrug, rapamycin,
paclitaxel, or a paclitaxel prodrug. The amphiphilic polymer may be
PEG-DSPE, PEG-PCL, or PEG-polyamino acid. A hydrophobic excipient may be
included, preferably, Vitamin E. A micelle composition may have a
concentration of between about 1 and about 50 mM, Vitamin E may have a
concentration of between about 2 and about 100 mM, and a rapamycin
concentration of between about 0.1 and about 10.0 mg/mL. A micelle
composition may also have the amphiphilic polymer concentration of
between about 3 and about 7 mM, the Vitamin E a concentration of between
about 8 and about 12 mM, and the rapamycin a concentration of between
about 0.3 and about 0.7 mg/ml. The ratio of Vitamin E to amphiphilic
polymer may be between about 0.2 and about 50 and the micelle may have a
diameter of less than about 200 nm. The ratio of rapamycin to polymer may
be about 0.1 and about 4.

[0148] A process for forming micelle compositions may comprise: mixing
amphiphilic polymer, hydrophobic excipient, and hydrophobic drug into an
organic solvent to form a solution and removing substantially all of the
solvent from the solution to leave a substantially solvent-free mixture.
The process may further include resuspending the substantially
solvent-free mixture in water or buffer. The process may also include
adding the solution to a substantially water solution before removing
substantially all of the solvent from the solution to leave a
substantially solvent-free mixture. The process for forming micelle
compositions may further include removing the drug that has not
incorporated into said micelle compositions. The process may be have the
mixing step be spinning the solution at between about 50 and about 1000
rpm.

[0149] As characteristics of the final aqueous solution, the amphiphilic
polymers may have a concentration of between about 0.1 mM and about 60
mM, and the hydrophobic excipients may have a concentration of between
about 0.1 mM and about 600 mM, and the drugs may have a concentration of
between about 0.1 mg/ml and about 10.0 mg/ml. Almost any organic solvent
may work in the process that all the components are soluble, for example,
but not exclusively, MeOH, acetone, THF, ACN. The solvent may be about a
50:50 chloroform:methane solution. Additionally, the spinning step and
the removing step of the process may occur simultaneously and the
resuspending step may be combined with ultrasonification for between
about 3 and about 20 minutes. The hydrophobic passenger drug may be
rapamycin, paclitaxel, paclitaxel prodrugs, geldanamycin, and
geldanamycin prodrugs.

[0150] A process for solubilizing rapamycin may comprise: dissolving
amphiphilic polymer, a hydrophobic excipient, and rapamycin into an
organic solvent to form a solution; mixing said solution; removing
solvent from said solution to form a substantially solvent-free
composition; and resuspending said substantially solvent-free mixture in
water or buffer. The resuspending step may form micelle compositions. The
polymers may be PEG-DSPE. A ratio of hydrophobic excipient to PEG-DSPE
may be between about 0.1 and about 3. The hydrophobic excipient may be
Vitamin E.

[0151] A micelle composition may comprise amphiphilic polymers and
geldanamycin. The micelle composition may also include a hydrophobic
excipient. The hydrophobic excipient may be Vitamin E. The geldanamycin
may be between about 200 and about 800 μg/ml.

[0152] A prodrug composition may have a log P o/w of at least about 3.5.
The prodrug may be of geldanamycin or paclitaxel. A geldanamycin prodrug
may have an amino spacer group at the C17 position, and an R group
adjacent said spacer group. The R group may be a carbon chain between
about 4 and about 24 carbons, more preferably between about 6 and about
16 carbons. The chain may be saturated or partially unsaturated. The R
group may be an ester, bromoester, aminoethyl-hexonate,
aminoethyl-dodeonate, aminoethyl-palmitate, aminoethyl-bromopalmitate, or
amino-hexadecyl. A micelle composition may comprise an amphiphilic
polymer and one of these geldanamycin prodrugs. The geldanamycin prodrug
may have a log Po/w of at least about 3.5.

[0153] A paclitaxel prodrug may have an amino linker group and an R group
adjacent said linker group. The amino linker group may be at the C7 or C2
position. The paclitaxel prodrug may have a log Po/w of at least about
3.5. The R group may be a carbon chain between about 4 and about 24
carbons, more preferably between about 6 and about 16 carbons. The chain
may be saturated or partially unsaturated. The R group may be an ester,
bromoester, aminoethyl-hexonate, aminoethyl-dodeonate,
aminoethyl-palmitate, aminoethyl-bromopalmitate, or amino-hexadecyl. A
micelle composition may comprise an amphiphilic polymer and one of these
paclitaxel prodrugs. The paclitaxel prodrug may have a log Po/w of at
least about 3.5.

[0154] A micelle composition may include a paclitaxel prodrug comprising
one of: 7-palmitate-paclitaxel, 7-palmitate-paclitaxel, 2-TBS-paclitaxel,
2-palmitate-paclitaxel, 2-TBS-7-palmitate-paclitaxel. A process for
forming the micelle compositions, may comprise: formulating a paclitaxel
prodrug having a log Po/w of at least about 3.5; mixing amphiphilic
polymer and said paclitaxel prodrug into an organic solvent to form a
solution; removing solvent from said solution to leave a substantially
solvent-free mixture; and resuspending said solvent-free mixture in water
or buffer. A process for forming micelle compositions may also comprise:
formulating a paclitaxel prodrug having a log Po/w of at least about 3.5;
mixing amphiphilic polymer and said paclitaxel prodrug into an organic
solvent to form a solution; removing solvent from said solution to leave
a substantially solvent-free mixture; and resuspending said solvent-free
mixture in water or buffer.

[0156] A process for forming micelle compositions with a paclitaxel
prodrug may comprise or produce: 7-palmitate-paclitaxel,
7-palmitate-paclitaxel, 2-TBS-paclitaxel, 2-palmitate-paclitaxel,
2-TBS-7-palmitate-paclitaxel.

[0157] A method of treatment for a disease or a condition in a human or an
animal comprising administering a micelle composition comprising an
amphiphilic polymer, a hydrophobic excipient and a hydrophobic passenger
drug. The hydrophobic passenger drug may be geldanamycin, geldanamycin
prodrugs, rapamycin, paclitaxel, or paclitaxel prodrugs. The amphiphilic
polymer may be PEG-DSPE, PEG-PCL, or PEG-polyamino acid. The hydrophobic
excipient may be Vitamin E. Human or animal diseases or conditions may:
cancer, neurological disorder, Alzheimer's disease, Huntington's disease,
restenosis, fungal infection, immunosuppression. The fungal infection may
be Candida albicans.

[0158] Although the invention has been described with reference to
preferred embodiments and examples thereof, the scope of the present
invention is not limited only to those described embodiments. As will be
apparent to persons skilled in the art, modifications and adaptations to
the above-described invention can be made without departing from the
spirit and scope of the invention, which is defined and circumscribed by
the appended claims. The following examples are provided for the intent
of illustrating embodiments and advantages of the invention and are not
intended to limit its scope.

Example 1

Formation of Micelles and Passenger Drugs

[0159] Doxorubicin and paclitaxel can be incorporated into micelle
compositions to be delivered to targeted tumors. PEG-poly(aspartic acid),
PEG-poly(aspartate), PEG-poly(lactide), PEG-DSPE are a few of the micelle
carriers that can encapsulate passenger drug compounds. See Table 1.

[0160] Loading of rapamycin into micelle compositions, which has a
solubility of 2.6 μg/ml in water. The loading efficiency of rapamycin
into PEG-DSPE increases proportionally with the increase of incorporated
tocopherol. The loading efficiency of rapamycin into PEG-PCL also
increases proportionally with the increase of incorporated tocopherol.
See Table 2.

Formation of Micelle Compositions with Incorporated Tocopherol and
Rapamycin

Dripwise Extraction Method of Forming Micelle Compositions

[0162] According to FIG. 19, amphiphilic polymers and the desired
passenger drug are dissolved in a highly water miscible solvent for which
they have excellent solubility. Examples include: MeOH, acetone, EtOH,
acetonitrile, THF, dioxane, and IPA.

[0163] For example to make a 0.5 ml solution of drug at 1 mg/ml and 2.5 mM
PEG-DSPE and 1:2 tocopherol:

[0164] Dissolve stated quantities of tocopherol, PEG-DSPE, and rapamycin
in 0.5 ml of acetone and load into a syringe. Use a syringe pump to
deliver the solution to solution of water at 25-50 μl/min (approx. 1
drop/10-15 s).

[0165] The volume of water should be sufficient so that the final solvent
to water ratio is 2:1 or less. Typically at least 1 ml of water should be
used.

[0166] The water (or other aqueous buffer [e.g. PBS]) is placed in a small
beaker with a stirbar, covered in parafilm, and placed on a stirplate
with vigorous stirring. Delivery is started and should finish in 15-45
minutes based upon the delivery rate.

[0167] For very hydrophobic polymers (e.g. PEG 5000:PCL 15000) a slower
flowrate (20 s/drop) may be used and for easily formed systems (e.g.
PEG-DSPE) the rate may be increased to 10 s/drop.

[0168] After delivery is done, the vial is placed under a stream of
nitrogen or other dry non-reactive gas (e.g. purified dry air, argon,
helium) and the solvent is evaporated. If necessary the solution can be
concentrated by the continuing the evaporation past the point that the
water is all gone. A benefit of using acetone verses azetrope forming
solvents (e.g. EtOH) is that all of the solvent can be removed under
these conditions. Also a solvent such as DMSO or DMF would not evaporate
before the water. In addition, the vial can be allowed to sit overnight
or longer (maybe without a purge gas) to allow the solvent to slowly
evaporate. This may be important for long hydrophobic chain polymers such
as the PEG-PCL that may swell in the presence of the acetone and would
require slow removal of the acetone to allow micelle stability.

[0169] After all of the organic is removed (and if the desired the
solution is further concentrated) the solution can be sterile filtered
(e.g. through a 0.2 μm or 0.45 μm syringe filter) to remove an
aggregates of unincorporated drug or other non-micelle, >200 nm sized
particles. Alternatively, the solution can be centrifuged to get rid of
aggregates of drugs. (e.g. 16000×g for 5 minutes).

[0170] Thin Film Evaporation Method of Forming Micelle Compositions.

[0171] Thin film evaporation method for forming micelle compositions
example is as follows: [0172] 1. Dissolve the desired passenger drug,
tocopherol, and amphiphilic polymer in a highly volative organic solution
in which they are soluble. See FIG. 18. [0173] 2. To make 1 ml of a final
5 mM of PEG-DSPE, 10 mM of tocopherol, 0.5 mg/ml rapamycin solution,
dissolve the components in a 10 ml 50:50 chloroform:MeOH solution. Place
in a 50-100 ml round bottom vacuum flask. Place flask on a rotary
evaporator, or rotovap, and spin at about 100 rpm and place under vacuum
to remove the solvent. It is important to control the vacuum so that the
solvent does not "bump" or violently evaporate/boil and backflow into the
rotovap condenser. [0174] 3. After all of the solvent is evaporated,
place under a very high vacuum (10-100 μbar) to remove all trace
solvent. This is especially important in the case of high tocopherol
loading because tocopherol is an oily viscous substance and the solvent
may be slow to evaporate from the tocopherol containing film. [0175] 4.
Add the appropriate volume of water or buffer. In this case 1 ml. Agitate
vigorously and the micelles will form. This can be assisted by
ultrasonification for 5-15 minutes.

[0176] According to FIG. 18, the loading efficiency of the drug increased
until the drug to amphiphilic unimer ratio reached 2:1. The loading
efficiency was about 40% of the desired rapamycin that was dissolved into
the volative solution. The loading efficiency of the desired rapamycin
then decreased after the drug:unimer ratio increased beyond 2:1 to a drug
loading efficiency of less than 20% at drug:unimer ratios of 3:1 and 4:1.
The PEG-DSPE micelle-tocopherol size may have been about 14±2 nm and
the micelle-tocopherol-rapamycin composition may have a size of about
16±2 nm. Thus, the rapamycin does not increase the micelle composition
to be beyond EPR standards.

Example 5

Rapamycin Incorporation into Micelle Compositions

[0177] The incorporation of rapamycin into the micelle compositions can be
detected by SEC. As shown in FIG. 24, the micelles and rapamycin both
come off the column at the same time, thus showing that they are
incorporated into one compound. Unincorporated amphyphylic unimers do not
form micelle compounds and come off the column at a later time. This
example was conducted in a Shodex 804 SEC column, at 0.75 ml/min, and 37
degrees C., and RI and 277 nm UV detection.

Example 6

Instability of PEG-DSPE Micelles Alone

[0178] As shown in FIG. 14, within a phosphate buffered saline solution,
PEG-DSPE micelles are very stable. When PEG-DSPE micelle compositions are
mixed in a phosphate buffered solution with 4% bovine serum albumin
(BSA), the micelle compositions are much less stable and the passenger
compound crashes out of the drug within 1 hour. The micelle compositions
were released into 37 degrees Celsius deionized water from a 7500
molecular weight cutoff dialysis.

[0179] As shown in FIG. 28, when micelle compositions are incorporated
with tocopherol, the compositions are more stable over time and the drugs
do not crash out. In the presence of 4% BSA, the 5 mM PEG-DSPE without
tocopherol crashed out within the first 20 hours, but the 5 mM PEG-DSPE
micelle composition with 10 mM tocopherol composition held together in
solution for almost 60 hours.

[0180] As shown in FIG. 16, about 60% of the micelle compositions stayed
intact for at least 25 hours.

Example 7

Core Rigidity of Micelle Compositions with Tocopherol

[0181] As shown to FIG. 13, the core viscosity, or rigidity, of a micelle
composition decreases slightly when tocopherol is incorporated. PEG-DSPE
without any tocopherol has a relative core viscosity of a little less
than about 3 Im/Ie. The core viscosity decreases when
tocopherol is added to the micelle composition. The core viscosity does
not decrease linearly, but holds steady at about 1 Im/Ie when
the PEG-DSPE:tocopherol ratio increases past 1:1. The decrease in micelle
composition core rigidity may decrease micelle stability and increase
drug diffusion.

[0182] As shown in FIG. 17, the core polarity of micelle compositions with
incorporated tocopherol molecules is lower than micelles without
tocopherol molecules. The core polarity of PEG-DSPE alone is about 1.1.
The core polarity of a PEG-DSPE and tocopherol micelle composition having
a PEG-DSPE:tocopherol ratio of 1:2 is about 0.8. The incorporation of
tocopherol may decrease core polarity and thereby increase the loading of
hydrophobic molecules. This will affect the release kinetics due to
enhanced partitioning.

Example 9

Increasing Size of Micelle Compositions with Tocopherol

[0183] The size of the micelle compositions is important because of the
extravasation into tumor site. The micelles should ideally be less than
about 400 nm in diameter in order to reach tumor sites. As shown in FIG.
24, the incorporation of tocopherol into micelle compositions does not
increase the size of the resulting micelle compositions beyond 400 nm in
diameter.

Example 10

Increasing Aggregate Number with Incorporation

[0184] As shown in FIG. 14, the aggregate number of polymers increases
with the incorporation of tocopherol into micelle compositions. The
increased aggregate number may indicate an enlarged core. The core
increased in size from 5 to 6 nm radius for the PEG-PCL 1:0 tocopherol to
the 1:20 tocopherol. The core increased from 1.5 nm to 3 nm radius for
the PEG-DSPE 1:0 tocopherol to the 1:2 tocopherol. At a
PEG-DPSE:tocopherol ratio of 1:0.5, then the difference in aggregate
numbers within the micelle composition becomes statistically significant.

Example 11

Rapamycin Loading by Diffusion-Evaporation

[0185] The weight percent of rapamycin in the micelle compositions when
there is tocopherol incorporated, showing the benefit of tocopherol
incorporation. As shown in FIG. 18, when there is no tocopherol
incorporated, at a rapamycin:micelle unimer ratio of 2:1, there is about
20 weight % rapamycin in the micelle composition. When there is either
1:1 or 1:2 PEG-DSPE:tocopherol ratios, then the weight % of rapamycin
increases past 25%.

Example 12

Tocopherol Effect on Rapamycin Release

[0186] As shown in FIG. 27, tocopherol increases the time over which
rapamycin is released in a polar buffer solution, but not significantly
so. The difference in drug retention between PEG-DSPE micelle without
tocopherol and PEG-DSPE with incorporated tocopherol is not statistically
significant.

[0187] As shown in FIG. 28, the effect of tocopherol on drug retention of
PEG-DSPE micelle compositions when in solution with 4% BSA is
statistically significant. 4% BSA is the concentration of albumin in the
human spinal cord. Tocopherol helps keep PEG-DSPE micelle compositions
stable in in vivo conditions for improved drug delivery.

[0188] As shown in FIG. 29, tocopherol increases the amount of rapamycin
and geldanamycin capable of being loaded into a PEG-PCL micelle. A
PEG-PCL:tocopherol ratio of 1:10 leads to a rapamycin load of 0.34 mg/ml.
That is at 90% loading efficiency. A 1:20 ratio of PEG-PCL to tocopherol
leads to a 54% loading efficiency of geldanamycin.

[0189] As shown in FIG. 30, tocopherol incorporation into PEG-PCL micelles
also help the resulting micelle composition retain rapamycin in 4% BSA
solution. This shows the stabilizing effect of tocopherol incorporation
into PEG-PCL micelles in in vivo conditions.

[0191] Male Sprague-Dawley rats (200-240 g) were obtained from Simonsen
Labs (Gilroy, Calif., USA) and given food (Purina Rat Chow 5001) and
water ad libitum in our animal facility for at least 3 days before use.
Rats were housed in temperature-controlled rooms with a 12 h light/dark
cycle. The day before the pharmacokinetic experiment the right jugular
veins of the rats were catherized with sterile silastic cannula (Dow
Corning, Midland, Mich., USA) under halothane anesthesia. This involved
exposure of the vessel prior to cannula insertion. After cannulation, the
Intramedic PE-50 polyethylene tubing (Becton, Dickinson and Company,
Franklin Lakes, N.J., USA) connected to the cannula was exteriorized
through the dorsal skin. The cannula was flushed with 0.9% saline. The
animals were transferred to metabolic cages and were fasted overnight.
Animal ethics approval was obtained from The Institutional Animal Care
and Use Committee at Washington State University.

[0192] Twelve male Sprague Dawley rats (average weight: 220 g) were
cannulated as described in the previous section. Each of the animals were
placed in separate metabolic cages, allowed to recover overnight, and
fasted for 12 h before dosing. On the day of experiment, the animals were
dosed intravenously with rapamycin (10 mg/kg) dissolved either in DMA,
PEG, and Tween 80 (control formulation), poly(ethylene
glycol)-β-poly((ε-caprolactone) (PEG-PCL formulation), or
PEG-PCL co-incorporated with α-tocopherol
(PEG-PCL+α-tocopherol formulation) (N=4 for each treatment group).
Serial blood samples (0.25 ml) were collected at 0, 1 min, 0.5, 1, 2, 4,
6, 12, 24, and 48 h. Each blood sample was divided into two 0.1 ml
fractions, the first one was collected into regular polypropylene
microcentrifuge tube and labeled as whole blood sample and stored at
-70° C. until analyzed. The second fraction was collected in
heparanized tubes (Monoject, Mansfield Mass.) and following
centrifugation, the plasma and red blood cell (RBC) fractions were
collected and stored at -70° C. until analyzed.

[0193] The protocol previously described by Annesley and Clayton, 2004 [1]
was slightly modified. For our purpose, 10 ul of whole blood, plasma,
calibrator or control was added in a regular polypropylene
microcentrigufe tube. Then, 250 ul of deionized water, 250 ul of aqueous
0.1 mol/L zinc sulfate, and 500 ul methanol containing the internal
standard were added. The mixture was vortexed for 30 seconds, and the
tubes were left at room temperature for 5-10 minutes. Then, the tubes
were centrifuged for 4 minutes, and the colorless supernatant was
analyzed. A 60 mg, 3 ml Oasis HLB column was utilized for the solid phase
extraction (SPE) clean up of the samples. The column was conditioned with
1 ml methanol followed by 1 ml of water. The prepared supernatant was
passed slowly through the column (1-2 ml/min), then the column was washed
with 1 ml of water and air-dried for about 30 seconds. The LC/MS analyses
were carried on a Agilent 1100 system. In the positive-ion mode the
monitored multiple-reaction monitoring transition (m/z) was: rapamycin
931.6→864.5. Separation was performed with a Waters Xtterra
MS18 2.1×100 mm maintained at 40° C. The injection
volume was 25 ul with a flow rate of 0.4 ml/min. The mobile phases were
(A) 10 mM ammonium acetate and 0.1% formic acid in water and (B) 10 mM
ammonium acetate and 0.1% formic acid in methanol. The gradient program
was 50% A and 50% B for the whole run (15 minutes).

[0194] Pharmacokinetic analysis was performed using WinNONLIN®
software (Ver. 1). Summary data were expressed as mean±standard error
of the mean (S.E.M.). The elimination rate constant (λn) was
estimated by linear regression of the plasma concentrations in the
log-linear terminal phase. The AUC0-∞ was calculated using the
combined log-linear trapezoidal rule for data from time of dosing to the
last measured concentration, plus the quotient of the last measured
concentration divided by λn. Non-compartmental pharmacokinetic
methods were used to calculate clearance (CL) and volume of distribution
(Vd) after iv dosing. The blood distribution of rapamycin was
calculated by dividing the rapamycin concentration detected in plasma by
the concentration detected in RBC at different time points after
intravenous dosing with the different rapamycin formulations.

[0195] Following intravenous administration of the rapamycin control
formulation, a small increase in rapamycin concentration was evident at
12 hours indicating the possibility of enterohepatic recycling (FIG. 1).
The total clearance of rapamycin was determined to be 1.12±0.14 L/h/kg
(Table 1). The volume of distribution of rapamycin is 20.94±3.65 L/kg,
which is greater than total body water, suggesting rapamycin is highly
distributed in tissue. The concentrations of rapamycin appeared to slowly
decline rapidly with a mean elimination half-life of 11.52±0.57 h. The
mean area under the curve (AUC), representing the total amount of drug
exposure in the blood over time, was 8.34±0.91 μgh/ml.

[0196] Following intravenous administration of the rapamycin PEG-PCl
formulation (FIG. 2), the total clearance of rapamycin was determined to
be 1.11±0.07 L/h/kg (Table 1). The volume of distribution of rapamycin
is 24.85±2.10 L/kg, which is greater than total body water, suggesting
rapamycin is highly distributed in tissue. The concentrations of
rapamycin appeared to decline slowly with a mean elimination half-life of
15.55±0.71 h. The mean area under the curve (AUC), representing the
total amount of drug exposure in the plasma over time, was 9.23±0.71
μgh/ml.

[0197] Following intravenous administration of the rapamycin PEG-PCl and
α-Tocopherol formulation (FIG. 3), the total clearance of rapamycin
was determined to be 0.84±0.03 L/h/kg (Table 1). The volume of
distribution of rapamycin is 17.74±1.27 L/kg, which is greater than
total body water, suggesting rapamycin is highly distributed in tissue.
The concentrations of rapamycin appeared to decline slowly with a mean
elimination half-life of 14.63±0.81 h. The mean area under the curve
(AUC), representing the total amount of drug exposure in the blood over
time, was 11.93±0.41 μgh/ml.

[0198] The plasma/RBC ratios were calculated at 1 min (FIG. 4) and 12
hours (FIG. 5) after intravenous dosing of the different rapamycin
formulations. The plasma/RBC ratios after 1 min and 12 hr i.v. dosing of
rapamycin control formulation are 2.21 and 0.41 respectively. The ratios
after i.v. dosing of rapamycin PEG-PC1 formulation are 3.44 and 0.48
respectively, and the rations after i.v. dosing of rapamycin
PEG-PCl+α-tocopherol are 4.80 and 0.76 respectively.

[0199] After i.v. dosing there was 40% mortality of the rats after the
rapamycin control formulation which occurred 0-2 hours after drug
administration. Control animals consistently appeared listless. There was
no mortality with either of the rapamycin micellular formulations. The
rats were held in metabolic cages and urine collected for 24 hour
intervals and volume measured. There was no difference in renal output
between groups.

[0200] Rapamycin pharmacokinetics has been studied extensively in
different species including rat, monkey, rabbit, and human. These studies
have characterized rapamycin to be a drug with a relatively long
half-life of more than 5 hours, with volume of distribution values that
indicates a substantial proportion of the drug residing extravascularly,
and rapidly absorbed in the body [2-5]. Rapamycin is a lipophilic
compound with a partition coefficient (XLogP) of 5.773 and is highly
distributed into the tissue as evidenced by the high volume of
distribution value. In addition, rapamycin is highly extracted as
suggested by its clearance values.

[0201] The different formulations studied show a change in the
pharmacokinetic parameters of rapamycin. There is a change in the volume
of distribution (Vd) of rapamycin from 20.94 L/kg in the control
formulation to 17.75 L/kg in the tocopherol formulation respectively.
Similarly the two formulations offer an increase in the half-life from
11.52 h (control) to 15.55 and 14.63 h for PEG-PCl and PEG-PCl+tocopherol
respectively. There is also an increase in AUC values and a decrease in
clearance values with the two formulations compared to the control. All
these pharmacokinetic parameter changes show an eventual higher residence
time of rapamycin in the body and increase in plasma residence suggests
less distribution into the RBC which may facilitate better distribution
to possible target sites, which eventually will exert a higher
pharmacological effect than the control formulation considering that all
the formulations were applied at the same dose (10 mg/kg). Thus, the
further study of the pharmacokinetic and pharmacodynamic effects of these
formulations is warranted.

[0202] The blood distribution of rapamycin was also studied in vivo, and
the plasma/RBC ratios were calculated at two time points (1 min and 12 h)
after intravenous dosing of the different rapamycin formulations. These
results show a higher distribution of rapamycin in plasma than red blood
cells at 1 minute in all the formulations. However, after 12 hours
rapamycin has a higher distribution in red blood cells than plasma. This
change in blood distribution among time could be explained by the fact
that rapamycin binds to FKBP [FK506 binding protein] in red blood cells
[6]. This protein binding could make the clearance of rapamycin out of
the red blood cells slower than the clearance out of the plasma giving
this biodistribution change. The two formulations (PEG-PCl and
PEG-PCl+tocopherol) at both time points (1 minute and 12 hours) show a
higher plasma/RBC ratio than the control formulation. This would
represent a higher concentration of rapamycin not bound to RBC proteins
making it more available to exert its pharmacological effects.

[0203] As shown in Table 27, geldanamycin prodrugs loaded into micelles
are pretty stable. Micelles loaded with
17-aminoethyl-palmitate-17-demethoxygeldanamycin or
17-aminoethyl-dodeconate-17-demethoxygeldnamycin release almost all the
drug after about 8 days. Micelles loaded with
17-aminoethyl-bromododeconate-17-demethoxygeldanamycin or
17-amino-hexyldecyl-17-demethoxygeldandamycin release substantially of
all the drug after about 12 days. Micelles loaded with
17-aminoethyl-bromohexonate-17-demethoxygeldanamycin or
17-aminoethyl-bromopalmitate-17-demethoxygeldanamycin release
substantially all the drug after about 14 days.

[0205] Synthesis of 7-palmitate-paclitaxel 4c. The method for synthesis of
2-palmitate-paclitaxel 4c is described infra. Synthesis of 4a-b were
according to the same procedure, with substitution of the appropriate
fatty anhydride.

[0207] 2-TBS-7-palmitate-paclitaxel 3. To a solution of 2 (50 mg, 0.053
mmol) in 1 ml dry toluene was added palmitic anhydride (38.3 mg, 0.0774
mmol). The reaction mixture was stirred at 90° C. for 18 h. The
resulting solution was washed with 1-M HCl (5 ml×1) followed by
water (5 ml×1), and the organic layer was dried over
Na2SO4. Removal of the solvent followed by preparatory TLC on
silica (1:1 EtOAc:hexane) provided 3 as a white solid (25 mg, 41% yield).

[0212] Paclitaxel prodrug loaded PEG-b-PCL micelles were prepared by
dissolving PEG-b-PCL (5000:10500, Mw/Mn 1.11, JCS Biopolytech
Inc., Toronto, Ontario Canada) and prodrug in a minimum volume of acetone
and adding drop-wise to vigorously stirred ddH2O using a syringe
pump. The organic solvent was then removed by stirring under an air
purge. Where stated, samples were further concentrated by prolonged
evaporation under an air purge. After removing the organic solvent,
PEG-b-PCL micelles were passed through a 0.22-μm polyestersulfone
filter to remove insoluble material and unincorporated drug [1]. In a
typical experiment, 1 μM of PEG-b-PCL was dissolved in 0.75 ml of dry
acetone and added dropwise (50 μL/min) to 2 ml of ddH2O yielding
0.5-mM PEG-b-PCL micelles after removing the volatile organic solvent.

[0214] PEG-b-PCL micelle prodrug release studies. Release experiments were
based on the methodology of Eisenberg and coworkers (Soo, P. L., et al.,
2002) with modifications for temperature and pH control. Micelle prodrug
solutions were prepared at 0.5 mM (PEG-b-PCL basis) with 20% w/w prodrug
as above, and 0.5 ml of each solution was diluted to 2.5 ml with
ddH2O and injected into 10000 MWCO dialysis cassettes (Pierce,
Rockford, Ill.) (n=4). Dialysis cassettes were placed in a well-mixed
temperature controlled water bath at 37° C., overflowed with
ddH2O so that the bath volume was refreshed every 15 to 20 min.
Peristaltic pumps under computer control separately injected 50-g/L
solutions of tribasic and monobasic phosphate to maintain pH at
7.4±0.05 (apparatus built in-house). At fixed time points, dialysis
cassette volumes were made up to 2.5 ml with ddH2O, 100-4 aliquots
withdrawn, and prodrug concentrations determined by reverse-phase HPLC
(see supra).

[0215] Diffusion constants and release half-lives were determined as
described previously by modeling release as Fickian diffusion from an
impenetrable sphere using the Crank solution for short time periods [1].
Linear regression of release data was performed in Sigma Plot 9.0
(Sysstat Software, Inc.). Diffusion constants were determined for
independent samples (n and reported as the average±standard deviation.
Release half-lives were determined using the calculated diffusion
constant in the Crank solution for 50% drug release.

[0216] Octanol-water partition coefficients. Octanol-water partition
coefficients (log Po/w) of paclitaxel prodrugs were determined
indirectly by microemulsion electrokinetic chromatography (MEEKC) based
on the technique of Klotz et al. (22). Running buffer was prepared by
titration of 25-mM sodium phosphate monobasic with 50-mM sodium
tetraborate to pH 7.00, and 1.44 g of sodium dodecyl sulfate, 6.49 g of
1-butanol, and 0.82 g of heptane were made up to 100 ml with
phosphate-borate buffer. The running buffer was ultrasonicated for 30 min
in a closed 250-ml flask in ice water (G112SP1 Special Ultrasonic
Cleaner, Laboratory Supplies Company Inc., Hicksville, N.Y.). Longer
times may be required to obtain a stable emulsion with lower power
ultrasonicators. Compounds and standards (n=3) were dissolved in the
running buffer (0.05 mg/ml) with 0.5 μL/ml of nitromethane and 0.5
μL/ml of 1-phenyldodecane by ultrasonication (10 min) in a closed tube
and centrifuged (16000×g, 3 min) to degas. A BioFocus 3000
capillary electrophoresis system (Bio-Rad, Hercules, Calif.) equipped
with a 50-μm ID×37-cm uncoated fused-silica column (Polymicron
Technologies LLC, Phoenix, Ariz.) was used for MEEKC experiments. The
column was prewashed with 1-M NaOH for 5 min and before runs with 0.1-M
NaOH for 1 min, ddH2O for 1 min, and running buffer for 1 min at 100
psi (690 kPa). Running conditions were 10 kV (ca. 30-35 μA, 30
min/run) at 20° C. with 1-psis injections (6.9 kPas) and detection
at 210 and 232 nm. Log Po/w and retention factors, k', were
calculated using the equations: